The pore of the sodium channel binds a variety of clinically-important drugs (local anesthetics, antileptics and antiarrhythmics). The overall goal of this proposal is to define, at a detailed molecular and biophysical level, the determinants of drug binding to voltage-dependent Na channels. Substantial progress has been made during the initial funding period using a combination of mutagenesis, electrophysiology and quantitative modelling. We have discovered major differences in lidocaine sensitivity between cardiac and skeletal muscle sodium channels, defined interactions between local anesthetic block and slow inactivation gating processes, and mapped the crucial determinants for high-affinity tetrodotoxin block in the outer pore. We now propose to continue functional characterization of normal and mutant channels while obtaining hard structural information on the Na channel pore and related drug binding sites. The central goals of the application are the following: 1) to elucidate the origin of the differences in lidocaine sensitivity between cardiac and skeletal muscle Na channels; 2) to define determinants of selectivity, conductance and drug block which lie outside the conventional selectivity filter region; 3) to determine the impact of pore flexibility on gating and drug block; 4) to test a model of the secondary structure in which a pore helix precedes the selectivity filter in each domain; and 5) to solubilize and purify the Na channel alpha subunit and/or a truncated protein containing the critical determinants of Na channel pore function and drug/toxin block. Given the central importance of sodium channels in normal physiology and in diseases of excitability (arrhythmias, epilepsy and myotonic disorders of muscle), this work promises not only to be of intrinsic biological interest but also of practical value in pharmacology.